ABSTRACT We are developing a combined modality therapeutic approach to eradicating metastatic cancers that are immunologically ?cold? and do not respond to immune checkpoint inhibition (ICI). Using an ?in situ vaccine? regimen consisting of 12 Gy focal external beam radiation therapy (EBRT) and intratumoral (IT) injection of tumor-specific antibody (mAb) + IL2, we have eradicated solitary, large, cold syngeneic tumors in mice. This in situ vaccine converts the targeted tumor into a focus of enhanced tumor antigen presentation resulting in increased T-cell infiltration and potent T-cell memory. However, the presence of an identical but untreated second tumor (2) on a mouse?s opposite flank inhibits the effect of this treatment, preventing eradication of the primary (1) tumor treated with EBRT + IT mAb-IL2. In this setting, the untreated 2 tumor causes tumor- specific immune unresponsiveness to EBRT + IT mAb-IL2 at the 1 tumor. We refer to this as concomitant immune tolerance (CIT). We can overcome CIT, and eliminate both tumors by giving IT mAb-IL2 to the 1 tumor and EBRT to both the 1 and 2 tumors. Delivering as little as 2 Gy EBRT to the 2 tumor can overcome CIT. Clinically, delivery of EBRT (even low dose) to all sites of metastatic disease is problematic, but this can effectively be achieved using molecular targeted radiation therapy (MTRT). MTRT is increasingly entering clinical oncology practice and our UW team has led preclinical and clinical testing of a novel class of MTRT using alkylphosphocholine (APCh) analogs that selectively deliver radiation to cancers in vivo. These show tumor-selective uptake in virtually all mammalian tumor cells and tumor locations tested (including > 90 tumor lines and in patients across various clinical trials). In a syngeneic murine melanoma model, we have observed a potent synergy between systemically administered ICI and MTRT delivered using our next-generation APC analog, 90Y-NM600. In a project that builds upon the ongoing collaborative progress of our multidisciplinary team, we will now systematically optimize the potency of combining MTRT with immunotherapy to enhance the immune response against immunologically cold tumors. In murine models, we will: 1) expand on preliminary data showing potent synergy with the combination of MTRT and ICI, 2) evaluate the capacity of MTRT to overcome CIT and enhance systemic anti-tumor immune response in the setting of multiple tumors where one is treated with in situ vaccine (EBRT + IT mAb-IL2) alone or in combination with ICI. Because murine models do not replicate the size and spatial distribution of human metastatic cancer and because these factors strongly influence the dosimetry of MTRT, we will test the immunomodulatory effects of MTRT + in situ vaccine in large breed companion canines (pet dogs) with naturally occurring metastatic melanoma. The insights and treatment regimens developed in these studies should enable rapid translation to clinical testing in patients and potentially for any type of metastatic cancer.